Acyl Carrier Proteins: The key to successfully engineering new biosynthetic pathways.

Polyketide natural products (produced by polyketide synthases (PKSs)) currently serve as a vast source of high value compounds with applications spanning agrochemicals, antibiotics, anti-cancer and human and vetinary medicine as well as tool compounds for use in biotechnological research. Enormous strides have been made in understanding the chemistry, biochemistry and structural biology of the PKSs that are complex collections of enzymes with a diverse array of architectures. For example in the biosynthesis of pseudomonic acid A, the main component of mupirocin, 64 different enzymes, some of which are organised into covalently linked subsets and others are free-standing. The evolution of the natural product via simple carbon building blocks derived from CoA (e.g. malonyl-CoA) can progress in a systematic, in-cis, pathway dictated by the linear arrangement of enzymes in the covalently linked modules but critically can recruit free-standing in-trans partners at critical junctures to perform additional chemistry. The most common protein within these assemblies, the acyl carrier protein (ACP), controls the passage of intermediates from one catalytic site of the PKS to the next (Figure 1) and may also encode motifs that can recruit trans-partners when required. They can also be arranged as di- or even tri-ACP repeats that adds an additional layer of complexity to their behaviour. These proteins are central to these PKSs but factors such as substrate specificity and protein-protein interactions remain poorly understood yet hold the key to successfully manipulating these pathways to produce high-value chemicals. We are internationally recognized as leaders in this field and recently we have used a multi-disciplinary approach combining chemistry, bioinformatics, molecular biology and NMR to propose a set of rules governing how ACPs specifically recruit a set of trans-acting enzymes to specifically incorporate b-branches at key stages of the molecular assembly of mupirocin (Haines et al., Nat. Chem. Biol. (2013) 9, 685-692). We will extend these studies to the structural biology of new ACPs, including tri-domain ACPs, to understand how these larger assemblies function and how they specifically recruit other trans-acting partners (e.g. enoyl-reductases). This will require the skills and expertise of both synthetic chemists (CLW), X-ray crystallography (PRR), NMR Spectroscopy (MPC) as well as bioinformatics (see collaborators). We will draw examples from the mupirocin and kalimantacin pathways, both of which contain examples of these ACPs and with up to 16 different ACPs per pathway, they provide model, self-contained orthogonal sets with which to test vital recognition control points.